The presence of donor specific alloantibodies (DSA) in allograft recipients portends a poor prognosis for the long-term survival of organ transplants. The main hypothesis of this proposal is that induction of robust donor specific transplantation tolerance will require establishment of humoral tolerance by eliminating alloreactive specificities from the primary B cell repertoire. It is a well-established fact that the B-cell compartment acquires tolerance to "self" antigens through clonal negative selection at early developmental checkpoints. This deletion of autoreactive cells occurs at the Immature and Transitional (TR) stages of B cell ontogeny. The stringency of clonal deletion at the TR`follicular (FO) B-cell developmental checkpoint is regulated by the dominant B lymphocyte survival factor, BLyS/BAFF. Therefore, it is logical to predict that recapitulating primary B-cell repertoire development in the presence of an allograft would cause negative selection of alloreactive clones from the FO compartment, yielding humoral transplantation tolerance. Here, we will determine whether this approach to primary B cell repertoire remodeling at the time of transplantation could promote robust humoral transplantation tolerance. Our preliminary studies have demonstrated two principle phenomena so far. First, that emergence of the B-Lymphocyte compartment in the presence of alloantigen leads to a striking absence of DSA, despite vigorous T-cell mediated acute allograft rejection. Second, in vivo neutralization of BLyS/BAFF depletes mature peripheral B-cell subsets and regulates the stringency of selection at the TR`FO checkpoint. Moreover, in vivo BLyS/BAFF neutralization, when combined with a transient course of Rapamycin, prevents acute rejection and causes donor specific transplantation tolerance to islet allografts. In this proposal, we will first determine whether de novo generation of the primary B-cell repertoire will prompt clonal deletion of alloreactive clones. Second, we will determine whether targeting BLyS for in vivo neutralization at the time of transplantation can lead to stringent clonal deletion of alloreactive B-cells, thereby yielding humoral transplantation tolerance. Finally, by utilizing a well-established model of chronic allograft vasculopathy, we will determine whether BLyS neutralization can prevent the progression of chronic rejection. These studies will provide the rationale for design of novel B- lymphocyte directed immunotherapeutics for the induction of donor specific transplantation tolerance. PUBLIC HEALTH RELEVANCE: The rejection of transplanted organs places a major burden on public health resources in the United States. Furthermore, the requirement for life-long immunosuppression to maintain transplant function not only poses a major cost burden on the health care system, but also imposes a dramatic degree of morbidity on the recipient. In this application, we propose a novel avenue of investigations into the possiblity of inducing donor specific immunological tolerance to allografts by targeting recipient B- lymphocytes.